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In vitro prototyping and rapid optimization of biosynthetic enzymes for cell design

Nature Chemical Biology volume 16, pages 912–919 (2020)Cite this article

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Abstract

The design and optimization of biosynthetic pathways for industrially relevant, non-model organisms is challenging due to transformation idiosyncrasies, reduced numbers of validated genetic parts and a lack of high-throughput workflows. Here we describe a platform for in vitro prototyping and rapid optimization of biosynthetic enzymes (iPROBE) to accelerate this process. In iPROBE, cell lysates are enriched with biosynthetic enzymes by cell-free protein synthesis and then metabolic pathways are assembled in a mix-and-match fashion to assess pathway performance. We demonstrate iPROBE by screening 54 different cell-free pathways for 3-hydroxybutyrate production and optimizing a six-step butanol pathway across 205 permutations using data-driven design. Observing a strong correlation (r = 0.79) between cell-free and cellular performance, we then scaled up our highest-performing pathway, which improved in vivo 3-HB production in Clostridium by 20-fold to 14.63 ± 0.48 g l−1. We expect iPROBE to accelerate design–build–test cycles for industrial biotechnology.

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Fig. 1: A two-pot cell-free framework for iPROBE.
Fig. 2: Individual pathway enzymes can be tuned in a pathway context and ranked using TREE scores with iPROBE.
Fig. 3: Enzymatic pathways can be screened with iPROBE to inform Clostridium expression for optimizing 3-HB production.
Fig. 4: Cell-free pathway testing combined with data-driven design of experiments quickly screens 205 unique pathway combinations and selects pathways for cellular butanol production.
Fig. 5: Clostridium fermentations show improved production of 3-HB and identification of a new route to 1,3-BDO.

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Data availability

All cell-free data generated and shown in this manuscript are provided in Supplementary Table 2 and Supplementary Datasets 1 and 2 (.xlsx). Any additional data or unique materials presented in the manuscript may be available from the authors upon reasonable request and through a materials transfer agreement.

References

  1. Nielsen, J. et al. Engineering synergy in biotechnology. Nat. Chem. Biol. 10, 319–322 (2014).

    CAS  PubMed  Google Scholar 

  2. Nielsen, J. & Keasling, J. D. Engineering cellular metabolism. Cell 164, 1185–1197 (2016).

    CAS  PubMed  Google Scholar 

  3. Green, E. M. Fermentative production of butanol–the industrial perspective. Curr. Opin. Biotechnol. 22, 337–343 (2011).

    CAS  PubMed  Google Scholar 

  4. Jones, D. T. & Woods, D. R. Acetone-butanol fermentation revisited. Microbiol. Rev. 50, 484–524 (1986).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Tracy, B. P., Jones, S. W., Fast, A. G., Indurthi, D. C. & Papoutsakis, E. T. Clostridia: the importance of their exceptional substrate and metabolite diversity for biofuel and biorefinery applications. Curr. Opin. Biotechnol. 23, 364–381 (2012).

    CAS  PubMed  Google Scholar 

  6. Takors, R. et al. Using gas mixtures of CO, CO2 and H2 as microbial substrates: the do’s and don’ts of successful technology transfer from laboratory to production scale. Microb. Biotechnol. 11, 606–625 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Charubin, K., Bennett, R. K., Fast, A. G. & Papoutsakis, E. T. Engineering Clostridium organisms as microbial cell-factories: challenges & opportunities. Metab. Eng. 50, 173–191 (2018).

    CAS  PubMed  Google Scholar 

  8. Connelly Jr., T. M. et al. Industrialization of Biology: A Roadmap to Accelerate the Advanced Manufacturing of Chemicals (The National Academies Press, 2015).

  9. Dudley, Q. M., Karim, A. S. & Jewett, M. C. Cell-free metabolic engineering: biomanufacturing beyond the cell. Biotechnol. J. 10, 69–82 (2015).

    CAS  PubMed  Google Scholar 

  10. Morgado, G., Gerngross, D., Roberts, T. M. & Panke, S. Synthetic biology for cell-free biosynthesis: fundamentals of designing novel in vitro multi-enzyme reaction networks. Adv. Biochem Eng. Biotechnol. 162, 117–146 (2018).

    CAS  PubMed  Google Scholar 

  11. Silverman, A. D., Karim, A. S. & Jewett, M. C. Cell-free gene expression: an expanded repertoire of applications. Nat. Rev. Genet. 21, 151–170 (2020).

    CAS  PubMed  Google Scholar 

  12. Bogorad, I. W., Lin, T. S. & Liao, J. C. Synthetic non-oxidative glycolysis enables complete carbon conservation. Nature 502, 693–697 (2013).

    CAS  PubMed  Google Scholar 

  13. Zhu, F. et al. In vitro reconstitution of mevalonate pathway and targeted engineering of farnesene overproduction in Escherichia coli. Biotechnol. Bioeng. 111, 1396–1405 (2014).

    CAS  PubMed  Google Scholar 

  14. Karim, A. S. & Jewett, M. C. A cell-free framework for rapid biosynthetic pathway prototyping and enzyme discovery. Metab. Eng. 36, 116–126 (2016).

    CAS  PubMed  Google Scholar 

  15. Dudley, Q. M., Anderson, K. C. & Jewett, M. C. Cell-free mixing of Escherichia coli crude extracts to prototype and rationally engineer high-titer mevalonate synthesis. ACS Synth. Biol. 5, 1578–1588 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Hold, C., Billerbeck, S. & Panke, S. Forward design of a complex enzyme cascade reaction. Nat. Commun. 7, 12971 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Kelwick, R. et al. Cell-free prototyping strategies for enhancing the sustainable production of polyhydroxyalkanoates bioplastics. Synth. Biol. 3, ysy016 (2018).

    CAS  Google Scholar 

  18. Kay, J. E. & Jewett, M. C. Lysate of engineered Escherichia coli supports high-level conversion of glucose to 2,3-butanediol. Metab. Eng. 32, 133–142 (2015).

    CAS  PubMed  Google Scholar 

  19. Karim, A. S., Heggestad, J. T., Crowe, S. A. & Jewett, M. C. Controlling cell-free metabolism through physiochemical perturbations. Metab. Eng. 45, 86–94 (2018).

    CAS  PubMed  Google Scholar 

  20. Dudley, Q. M., Nash, C. J. & Jewett, M. C. Cell-free biosynthesis of limonene using enzyme-enriched Escherichia coli lysates. Synth. Biol. 4, ysz003 (2019).

    CAS  Google Scholar 

  21. Grubbe, W. S., Rasor, B. J., Krüger, A., Jewett, M. C. & Karim, A. S. Cell-free styrene biosynthesis at high titers. Preprint at bioRxiv https://doi.org/10.1101/2020.03.05.979302 (2020).

  22. Karim, A. S. & Jewett, M. C. Cell-free synthetic biology for pathway prototyping. Methods Enzymol. 608, 31–57 (2018).

    PubMed  Google Scholar 

  23. Clomburg, J. M., Crumbley, A. M. & Gonzalez, R. Industrial biomanufacturing: the future of chemical production. Science 355, 6320 (2017).

    Google Scholar 

  24. Tseng, H. C., Martin, C. H., Nielsen, D. R. & Prather, K. L. Metabolic engineering of Escherichia coli for enhanced production of (R)- and (S)-3-hydroxybutyrate. Appl. Environ. Microbiol. 75, 3137–3145 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. McMahon, M. D. & Prather, K. L. Functional screening and in vitro analysis reveal thioesterases with enhanced substrate specificity profiles that improve short-chain fatty acid production in Escherichia coli. Appl. Environ. Microbiol. 80, 1042–1050 (2014).

    PubMed  PubMed Central  Google Scholar 

  26. Jewett, M. C. & Swartz, J. R. Mimicking the Escherichia coli cytoplasmic environment activates long-lived and efficient cell-free protein synthesis. Biotechnol. Bioeng. 86, 19–26 (2004).

    CAS  PubMed  Google Scholar 

  27. Jewett, M. C., Calhoun, K. A., Voloshin, A., Wuu, J. J. & Swartz, J. R. An integrated cell-free metabolic platform for protein production and synthetic biology. Mol. Syst. Biol. 4, 220 (2008).

    PubMed  PubMed Central  Google Scholar 

  28. Woolston, B. M., Emerson, D. F., Currie, D. H. & Stephanopoulos, G. Rediverting carbon flux in Clostridium ljungdahlii using CRISPR interference (CRISPRi). Metab. Eng. 48, 243–253 (2018).

    CAS  PubMed  Google Scholar 

  29. Fluchter, S. et al. Anaerobic production of poly(3-hydroxybutyrate) and its precursor 3-hydroxybutyrate from synthesis gas by autotrophic Clostridia. Biomacromolecules 20, 3271–3282 (2019).

    PubMed  Google Scholar 

  30. Atsumi, S. et al. Metabolic engineering of Escherichia coli for 1-butanol production. Metab. Eng. 10, 305–311 (2008).

    CAS  PubMed  Google Scholar 

  31. Inui, M. et al. Expression of Clostridium acetobutylicum butanol synthetic genes in Escherichia coli. Appl. Microbiol. Biotechnol. 77, 1305–1316 (2008).

    CAS  PubMed  Google Scholar 

  32. Shen, C. R. et al. Driving forces enable high-titer anaerobic 1-butanol synthesis in Escherichia coli. Appl. Environ. Microbiol. 77, 2905–2915 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Nguyen, N. P., Raynaud, C., Meynial-Salles, I. & Soucaille, P. Reviving the Weizmann process for commercial n-butanol production. Nat. Commun. 9, 3682 (2018).

    PubMed  PubMed Central  Google Scholar 

  34. Li, F. et al. Coupled ferredoxin and crotonyl coenzyme A (CoA) reduction with NADH catalyzed by the butyryl-CoA dehydrogenase/Etf complex from Clostridium kluyveri. J. Bacteriol. 190, 843–850 (2008).

    CAS  PubMed  Google Scholar 

  35. Qi, F. et al. Improvement of butanol production in Clostridium acetobutylicum through enhancement of NAD(P)H availability. J. Ind. Microbiol. Biotechnol. 45, 993–1002 (2018).

    CAS  PubMed  Google Scholar 

  36. Köpke, M. et al. Clostridium ljungdahlii represents a microbial production platform based on syngas. Proc. Natl Acad. Sci. USA 107, 13087–13092 (2010).

    PubMed  Google Scholar 

  37. Chowdhury, N. P., Kahnt, J. & Buckel, W. Reduction of ferredoxin or oxygen by flavin-based electron bifurcation in Megasphaera elsdenii. FEBS J. 282, 3149–3160 (2015).

    CAS  PubMed  Google Scholar 

  38. Gao, H. J., Wu, Q. & Chen, G. Q. Enhanced production of D-(−)-3-hydroxybutyric acid by recombinant Escherichia coli. FEMS Microbiol. Lett. 213, 59–65 (2002).

    CAS  PubMed  Google Scholar 

  39. Yun, E. J. et al. Production of (S)-3-hydroxybutyrate by metabolically engineered Saccharomyces cerevisiae. J. Biotechnol. 209, 23–30 (2015).

    CAS  PubMed  Google Scholar 

  40. Liew, F. et al. Metabolic engineering of Clostridium autoethanogenum for selective alcohol production. Metab. Eng. 40, 104–114 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Perez, J. M., Richter, H., Loftus, S. E. & Angenent, L. T. Biocatalytic reduction of short-chain carboxylic acids into their corresponding alcohols with syngas fermentation. Biotechnol. Bioeng. 110, 1066–1077 (2013).

    CAS  PubMed  Google Scholar 

  42. Kataoka, N. et al. Enhancement of (R)-1,3-butanediol production by engineered Escherichia coli using a bioreactor system with strict regulation of overall oxygen transfer coefficient and pH. Biosci. Biotechnol., Biochem. 78, 695–700 (2014).

    CAS  Google Scholar 

  43. Nemr, K. et al. Engineering a short, aldolase-based pathway for (R)-1,3-butanediol production in Escherichia coli. Metab. Eng. 48, 13–24 (2018).

    CAS  PubMed  Google Scholar 

  44. Jing, F. et al. Direct dehydration of 1,3-butanediol into butadiene over aluminosilicate catalysts. Catal. Sci. Technol. 6, 5830–5840 (2016).

    CAS  Google Scholar 

  45. Heijstra, B. D., Kern, E., Koepke, M., Segovia, S. & Liew, F. M. Novel bacteria and methods of use thereof. US patent 20130217096A1 (2013).

  46. Liew, F. et al. Gas fermentation: a flexible platform for commercial scale production of low-carbon fuels and chemicals from waste and renewable feedstocks. Front. Microbiol. 7, 694 (2016).

    PubMed  PubMed Central  Google Scholar 

  47. Heap, J. T., Pennington, O. J., Cartman, S. T. & Minton, N. P. A modular system for Clostridium shuttle plasmids. J. Microbiol. Methods 78, 79–85 (2009).

    CAS  PubMed  Google Scholar 

  48. Nagaraju, S., Davies, N. K., Walker, D. J., Kopke, M. & Simpson, S. D. Genome editing of Clostridium autoethanogenum using CRISPR/Cas9. Biotechnol. Biofuels 9, 219 (2016).

    PubMed  PubMed Central  Google Scholar 

  49. Köpke, M. et al. 2,3-Butanediol production by acetogenic bacteria, an alternative route to chemical synthesis, using industrial waste gas. Appl. Environ. Microbiol. 77, 5467–5475 (2011).

    PubMed  PubMed Central  Google Scholar 

  50. Kwon, Y. C. & Jewett, M. C. High-throughput preparation methods of crude extract for robust cell-free protein synthesis. Sci. Rep. 5, 8663 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Jewett, M. C. & Swartz, J. R. Substrate replenishment extends protein synthesis with an in vitro translation system designed to mimic the cytoplasm. Biotechnol. Bioeng. 87, 465–472 (2004).

    CAS  PubMed  Google Scholar 

  52. Koepke, M., Jensen, R. O., Behrendorff, J. B. Y. H. & Hill, R. E. Genetically engineered bacterium comprising energy-generating fermentation pathway. US patent 9,738,875 (2017).

  53. Valgepea, K. et al. Maintenance of ATP homeostasis triggers metabolic shifts in gas-fermenting acetogens. Cell Syst. 4, 505–515 (2017).

    CAS  PubMed  Google Scholar 

  54. Haykin, S. Neural Networks: A Comprehensive Foundation (Prentice Hall PTR, 1994).

  55. Whitley, D. A genetic algorithm tutorial. Stat. Comput. 4, 65–85 (1994).

    Google Scholar 

  56. Nelder, J. A. & Mead, R. A simplex method for function minimization. Computer J. 7, 308–313 (1965).

    Google Scholar 

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Acknowledgements

We thank A. M. Mueller, R. T. Tappel, W. Allen, L. Tran and S. D. Brown (LanzaTech) for conversations regarding this work. In addition, we thank C. Reynolds (Lockheed Martin) for conversations on the design of experiments using neural networks. This work is supported by the US Department of Energy, Office of Biological and Environmental Research in the Department of Environment Office of Science under award number DE-SC0018249. M.C.J. gratefully acknowledges the David and Lucile Packard Foundation and the Camille Dreyfus Teacher–Scholar Program. We also thank the following investors in LanzaTech’s technology: BASF, CICC Growth Capital Fund I, CITIC Capital, Indian Oil Company, K1W1, Khosla Ventures, the Malaysian Life Sciences, Capital Fund, L. P., Mitsui, the New Zealand Superannuation Fund, Petronas Technology Ventures, Primetals, Qiming Venture Partners, Softbank China and Suncor.

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Authors and Affiliations

  1. Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL, USA

    Ashty S. Karim, Quentin M. Dudley, Samantha A. Crowe, Jacob T. Heggestad, William S. Grubbe, Blake J. Rasor & Michael C. Jewett

  2. Chemistry of Life Processes Institute, Northwestern University, Evanston, IL, USA

    Ashty S. Karim, Quentin M. Dudley, Samantha A. Crowe, Jacob T. Heggestad, William S. Grubbe, Blake J. Rasor & Michael C. Jewett

  3. Center for Synthetic Biology, Northwestern University, Evanston, IL, USA

    Ashty S. Karim, Quentin M. Dudley, Samantha A. Crowe, Jacob T. Heggestad, William S. Grubbe, Blake J. Rasor & Michael C. Jewett

  4. LanzaTech Inc., Skokie, IL, USA

    Alex Juminaga, Yongbo Yuan, Shivani Garg, Tanus Abdalla, FungMin (Eric) Liew, Amy Quattlebaum, Rasmus O. Jensen, Sean D. Simpson & Michael Köpke

  5. Lockheed Martin Advanced Technology Laboratories, Cherry Hill, NJ, USA

    David N. Coar, Maria Torculas, Michael Krein & Jeffrey A. Stuart

  6. Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Chicago, IL, USA

    Michael C. Jewett

  7. Simpson Querrey Institute, Northwestern University, Chicago, IL, USA

    Michael C. Jewett

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  1. Ashty S. Karim
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Contributions

A.S.K., S.D.S., M.K. and M.C.J. designed the study. A.S.K., Q.M.D. and M.C.J. developed the cell-free framework. A.S.K., S.A.C., J.T.H., W.S.G. and B.J.R. performed all cell-free experiments. A.S.K. and Q.M.D. analyzed cell-free data. A.J. performed Clostridium strain engineering for 3-HB and 1,3-BDO. T.A. performed C. autoethanogenum gas fermentation for 3-HB and 1,3-BDO. Y.Y., F.E.L., R.O.J., S.G. and M.K. performed C. autoethanogenum strain engineering and gas fermentation for butanol. A.J., Y.Y. and M.K.. analyzed C. autoethanogenum data. A.Q. developed analytical methods for 3-HB, 1,3-BDO and butanol. D.C., M.T., M.Kr. and J.S. performed all design of experiments using neural networks. A.S.K., M.K. and M.C.J. wrote the manuscript.

Corresponding authors

Correspondence to Michael Köpke or Michael C. Jewett.

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Competing interests

A.J., T.A., S.G., A.Q., Y.Y., F.E.L., R.O.J., S.D.S. and M.K. are employees of LanzaTech, which has commercial interest in gas fermentation with C. autoethanogenum. Production of 3-HB, 1,3-BDO and 1-butanol from C1 gases has been patented (US patents 9,738,875 and 9,359,611). A.S.K. and M.C.J. are co-inventors on the US provisional patent application 62/173,818 that incorporates discoveries described in this manuscript. All other authors declare no competing interests.

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Supplementary information

Supplementary Information

Supplementary Tables 1 and 2, Figs. 1–11 and Notes 1 and 2.

Reporting Summary

Supplementary Dataset 1

The dataset contains five sheets related to the 3-HB data presented in this manuscript. Sheet 1 provides enzyme nomenclature, sheet 2 lists enzyme combinations, sheet 3 lists enzyme concentrations in final reactions, sheet 4 lists metabolite concentrations over time and sheet 5 lists all TREE scores and component parts.

Supplementary Dataset 2

The dataset contains five sheets related to the butanol data presented in this manuscript. Sheet 1 provides enzyme nomenclature, sheet 2 lists enzyme combinations, sheet 3 lists enzyme concentrations in final reactions, sheet 4 lists metabolite concentrations over time and sheet 5 lists all TREE scores and component parts.

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Karim, A.S., Dudley, Q.M., Juminaga, A. et al. In vitro prototyping and rapid optimization of biosynthetic enzymes for cell design. Nat Chem Biol 16, 912–919 (2020). https://doi.org/10.1038/s41589-020-0559-0

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